Naturally occurring cyclosporins are poly-N-methyl, cyclic undecapeptides, isolated from fungi. Cyclosporin A has an immunosuppressive activity and has been used for almost 40 years to prevent rejection in kidney, heart and liver transplant recipients. It has anti-inflammatory property and is useful for treating rheumatoid arthritis, severe psoriasis, Behget's uveitis and dry eye disease. In addition, it is useful for treating severe ulcerative colitis, Crohn's disease, alopecia greata, aplastic anemia, HSV-1 stromal keratitis, systemic lupus erythematosus, and severe lupus nephritis.
The anti-HIV activity of cyclosporin A was discovered (Klatzmann, D., et al., 1986, C R Acad. Sci. III, 303(9):343-8; Wainberg, M. A., et al., 1988, Blood, 72, 1904-10; Luban, J., et al., 1993, Cell, 73, 1067-1078; each of which is incorporated herein by reference). Its non-immunosuppressive derivative, NIM-811 was reported to have potent anti HIV activity, due to its ability to inhibit cyclophilin A (Franke, E. K., et al., 1994, Nature, 372, 359-362; Thali, M., et al., 1994, Nature, 372, 363-365; Gamble, T. R., et al., 1996, Cell, 87, 1157-1159; Rosenwirth B., et al., 1994, Antimicrob. Agents Chemother., 38, 1763-1772; each of which is incorporated herein by reference).
Cyclosporin A and its non-immunosuppressive derivatives, as such as NIM-811 (N-MeIle-4-Cyclosporin), Debio-025, and SCY-635, inhibit cyclophilin A and B, which interact with HCV protein NS5B and stimulate its RNA-binding activity. As a result, these compounds have an effective anti-HCV activity (Watashi, K., et al., 2007, Rev. Med. Virol., 17:245-252.37; Inoue, K., et al., 2001, Nippon Rinsho., 59, 1326-30; Inoue, K., et al., 2003, J. Gastroenterol., 38, 567-72; Watashi, K., et al., 2003, Hepatology, 38, 1282-8; each of which is incorporated herein by reference). Currently, NIM-811, Debio-025, and SCY-635 are undergoing clinical trials for treating HCV.
NIM-811 and Debio-025 have a chemical structure similar to cyclosporine A, and have poor pharmacokinetic profile and poor oral absorption. In addition, they are metabolized by P450 for inducing drug interactions (Lill, J., et al., 2000, Am J Health-Syst Pharm 57, 1579; incorporated herein by reference).
SCY-635 has an improved pharmacokinetic profile and low blood serum binding. In addition, it is less metabolized by P450 and has low potential for drug-drug interactions. SCY-635's in vitro anti-HCV activity (EC50) was reported to be 0.10 μM, by using the luciferase end point method, by Hopkins, S. et al., 2010, Antimicrob. Agents Chemother., 54, 660-672, incorporated herein by reference. However, SCY-635 is not chemically stable according to testing results in our laboratory. SCY-635 is easily converted to its diastereoisomer by epimerization, which is expected to have poor binding activity with cyclophilin, and therefore has poor anti-viral activity.
Cyclosporin A and its non-immunosuppressive derivatives were also found to possess anti-HBV activity through the inhibition of cyclophilins (Chokshi, S., et al., 2011, Abstract 190 (Poster Presentations), 46th Annual Meeting of the European Association for the Study of the Liver (EASL 2011), Berlin, March 30-April 3; Tian, X. C., et al., 2010, J. Virol., 84, 3373-3381; Xia, W. L., et al., 2004, Hepatobiliary Pancreat Dis Int., 4, 18-22; Michael, J., et al., 2003, J. Virol., 77, 7713-7719; each of which is incorporated herein by reference).
Furthermore, Cyclophilin were reported to regulate life cycles and pathogenesis of several viruses, including influenza A virus, severe acute respiratory syndrome coronavirus, and vaccinia virus (Castro, A. P., et al., 2003, J. Virol., 77, 9052-9068; Chen, Z., L., et al., 2005, J. Infect. Dis. 191, 755-760; Liu, X. L., et al., 2009, Cell Microbiol., 11, 730-741; each of which is incorporated herein by reference). Cyclosporin A and its non-immunosuppressive derivative also possess such anti viral-activities.
N-MeVal-4-Cyclosporin (SDZ 220-384), another non-immunosuppressive cyclosporine derivative, has similar chemical structure and similar biological activity compared to NIM-811 (Flirt, H., et al., 1993, Ann. N Y Acad Sci. 696, 47-53; Zenke, G., et al., 1993, Ann N Y Acad. Sci. 23; 685:330-5).
Hepatitis C virus (HCV) is a small (55-65 nm in size), enveloped, positive sense single strand RNA virus in the family Flaviviridae. HCV has a high rate of replication and has an exceptionally high mutation rate. Most people infected with HCV (about 80%) develop chronic, persistent infection. More than 4 million Americans have been infected with HCV and more than 200 million people are estimated to be infected chronically worldwide. About 35,000 new cases of hepatitis C are estimated to occur in the United States each year. HCV infection is responsible for about 50% of all chronic liver disease, 30% of all liver transplants, and 30% of all cirrhosis, end-stage liver disease, and liver cancer in the U.S. The peg-interferon and ribavirin combination is the standard treatment for chronic hepatitis C but has low efficacy against HCV infection. Recently, the FDA has approved Vertex's Incivek (telaprevir) and Merck's Victrelis (boceprevir) as an add-on to the current interferon/ribavirin therapy for treating HCV. Both drugs are HCV protease inhibitors and target virus to prevent its replication. However, due to the fast mutation of HCV, drug resistance can be developed in a short period of time for the new drugs. There exists a need for an effective therapeutic for HCV treatment.
Hepatitis B virus (HBV) is a 42 nm partially double stranded DNA virus, composed of a 27 nm nucleocapsid core (HBcAg), surrounded by an outer lipoprotein envelope containing the surface antigen (HBsAg). About a quarter of the world's population, more than 2 billion people, have been infected with the hepatitis B virus. This includes 350 million chronic carriers of the virus. The disease has caused epidemics in parts of Asia and Africa, and it is endemic in China. Chronic hepatitis B will cause liver cirrhosis and liver cancer-a fatal disease with very poor response to current chemotherapy. Although the infection is preventable by vaccination and HBV load and replication can be reduced by current antiviral drugs lamivudine (Epivir), adefovir (Hepsera), tenofovir (Viread), telbivudine (Tyzeka) and entecavir (Baraclude) and the two immune system modulators interferon alpha-2a and PEGylated interferon alpha-2a (Pegasys), none of the available drugs can clear the infection. There remains a need for an effective therapeutic for treating or preventing HBV infection.
The non-immunosuppressive Cyclosporins derivatives bind to cyclophilin, a family of host proteins that catalyze cis-tans peptidyl-prolyl isomerization in protein folding, which is crucial for the processing, maturation of the viral proteins for viral replication. It is also different to current anti-HIV and anti-HCV drugs, the advantages of targeting host cofactors—cyclophilins by cyclosporine derivatives is the presumed higher genetic barrier to development of resistance (Rosenwirth, B., et al., 1994, Antimicrob. Agents Chemother., 38, 1763-1772; Tang, H. L. et al., 2010, Viruses, 2, 1621-1634; Hopkins, S. et al., 2010, Oral Presentation, Scynexis's SCY-635 Demonstrates Impressive Barrier to Resistance in HCV Treatment, the 45th Annual Meeting of the European Association for the Study of the Liver (EASL 2010), Vienna, Austria, April 14-18; each of which is incorporated herein by reference). Cyclosporine derivatives affect a new target-cyclophilin, and therefore represent a new mechanism of action against HCV viruses.
Cyclophilins are a family of enzymes that assist in the folding and transportation of other proteins synthesized within a cell. Protein folding or misfolding plays a important role in the pathophysiology of a number of serious diseases, such as viral diseases (HIV, HBV, HCV, and herpes simplex virus), central nervous system disorders (mitochondrial protection for stroke, traumatic brain and spinal cord injury, Alzheimer, Parkinson's Disease, and Huntington's Diseases), cancer, cardiovascular diseases (reperfusion injury, heart attack, chronic heart failure), inflammation (respiratory inflammation, asthma, ulcerative colitis, rheumatoid arthritis, dry eye disease), muscular dystrophy, Atopic Dermatitis, anti fungal and anti-parasitic treatment, and hair growth. Cyclosporin derivatives target cyclophilin and can play a crucial role for treatment of such many diseases.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.